Multiple signaling molecules, including BMPs, FGFs, Shh, and Wnt proteins, have been implicated in mediating tissue interactions that regulate tooth initiation, morphogenesis and differentiation. In mice, the odontogenic potential, defined as the capability of an odontogenic tissue to elicit tooth developmental program in non-dental tissues, resides initially in dental epithelium, and then shifts to dental mesenchyme. Our long-term goal is to delineate the molecular basis of the odontogenic potential. Based on our previous studies, we hypothesize that during tooth initiation, BMP4 functions as an essential component of the odontogenic potential in the early dental epithelium, to induce the formation of the basal layer epithelium as well as to stimulate local cell proliferation to form a tooth bud. We further hypothesize that Wnt signaling pathway constitutes a component of the odontogenic potential in dental mesenchyme. Two specific aims are proposed to test these hypotheses. In Aim 1, the role of BMP4 in the early dental epithelium in tooth initiation will be defined by conditional knockout studies via the Cre/LoxP approach to achieve tissue-specific mutation of Bmp4 in the dental epithelium. This approach will also be used to delete the Bmpr-lA and Bmpr-IB genes in the dental epithelium to study if BMP4 acts intra-epithelially. Whether signaling of BMP4 to dental mesenchyme is essential for the acquisition of odontogenic potential in the mesenchyme will be tested by tissue recombination studies. Furthermore, whether Shh mediates Bmp4 activity in dental epithelium will be examined by conditional knockout and transgenic rescue experiments. In Aim 2, if Wnt signaling acts as a critical component of the odontogenic potential in dental mesenchyme will be studied by disrupting Writ signaling pathway in tissues used for tissue recombination. This will be achieved by overexpressing Wnt antagonist sFrp3 in dental mesenchyme, and Dkk1 and a dominant negative form of Tcf in responding non-dental epithelia via microelectroporation and virus-mediated gene delivery. The potential role of Wnt5a in the induction of tooth formation by dental mesenchyme will also be tested. Lastly, whether different sFrps confer the different inductive properties to incisor and molar mesenchyme will be determined by virus-mediated gene overexpression and RNAi-mediated gene knock-down studies, together with tissue recombination and organ culture studies.